The tomb contains the family of Lisa Gherardini, a silk merchant's wife who is believed to have posed for the artist's famous 16th century masterpiece.
Scientists cut a round hole in the stone church floor above the family crypt of Florentine silk merchant Francesco del Giocondo. The tomb lies behind the altar of the Santissima Annunziata Basilica in Italy, 'BBC News' reported.
The DNA extracted from bones in the tomb will be compared with that of three female skeletons that were found buried last year at a nearby convent. One of the skeletons is believed to be that of Gherardini.
"When we find a match between mother and child - then we will have found the Mona Lisa," said researcher Silvano Vinceti.
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He added that once a DNA match is made, an image of Gherardini's face can be generated from the skull and compared with the painting.
Gherardini became a nun after her husband's death. She died at the age of 63 at the Convent of Saint Ursula on July 15, 1542.
On display at the Louvre museum in Paris, 'Mona Lisa's' smile has baffled historians for centuries. One popular theory even suggests it was a self-portrait by Leonardo.